Method for producing a nanostructured complex (CFI-1), a protein-associated nanostructured complex (MRB-CFI-1) and use

ABSTRACT

Disclosed is a method of obtaining a protein-associated nanostructured complex (MRB-CFI-1) by chemical synthesis and antitumor use. The main use is in treating cancer, both in animals and humans. The complex has singular antitumor activity, and can potentially be used as a substitute and/or act as an adjuvant for other commercial antineoplastic drugs.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 16/617,493, filed Nov. 26, 2019, the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention refers to a process of obtaining an inorganicnanostructured complex (CFI-1), a protein-associated nanostructuredcomplex (MRB-CFI-1) and use.

The main technology application is the treatment of urinary bladdercancer, both in animals and in humans. Its antitumor activity is uniqueand a potentially substitute for other commercial antineoplastic drugs.

BACKGROUND OF THE INVENTION

All organs of the urogenital tract are potential spots for malignanttumors. The incidence and type vary from organ to organ. Urinary bladdercancer (CB) represents the second most common malignant disease of theurinary tract (Siegel et al., 2012; American Cancer Society, 2016).

The American Cancer Society estimated about 76,960 new cases of CB in2016 in the United States, being 58,950 in men and 18,010 in women. Theestimate also predicted 16,390 deaths due to CB, being 11,820 in men and4,570 in women (American Cancer Society, 2016). According to data fromthe National Cancer Institute (INCA, 2016), the estimate for Brazil in2016 was 9,670 new cases of CB, being 7,200 in men and 2,470 in women.In 2013, 3,642 deaths were reported due to CB, being 2,542 in men and1,099 in women, thus demonstrating a drastic increase in the prevalenceof this type of tumor.

More than 70% of the incidence of CB is superficial (pTis, pTa and pT1),of non-invasive tumor (CBNMI), and the occurrence of an invasive diseaseis occasional (Askeland et al., 2012). However, 50% of invasivenon-muscle tumors recurrence to 4 years after treatment and 11% evolveto the invasive phenotype (Askeland et al., 2012).

The histological staging of CB is determined by the depth of tumorinvasion of the bladder wall and will depend on the transurethralresection (RTU) of the tumor, by endoscopic approach, for a correctdiagnosis. Fragments of superficial and deep resection should beanalyzed separately (Epstein et al., 1998; Epstein, 2003). The TumourNode Metastasis (TNM) classification 2009 (UICC—Union for CancerControl) is used for staging.

A significant number of risk factors have been related to thedevelopment of CB. According to record data from the INCA populationdatabase, the greatest risk factor for the development of CB is smoking,accounting for about 66% of new cases in men and 30% in women (INCA,2016). In the meta-analysis of epidemiological studies of Zeegers et al.(2000) on the impact of smoking characteristics on the risk of urinarytract cancer, smoking was appointed as a factor that substantiallyincreases the risk for the development of bladder cancer. Cigaretteshave dozens of toxic substances, including aromatic amines and N-nitrousanalogues of MNU (N-methyl-N-nitrosourea), a potent carcinogen.

Another potential risk factor for the development of CB is occupationalexposure to aromatic amines by workers from rubber, textile and inkindustries, and infection by Schistosoma haematobiun, that is endemic inMediterranean countries, such as Egypt (Zeegers et al., 2000; Poon etal., 2015; Rosenquist & Grollman, 2016). Exposure to certain substancessuch as arsenic, which may be present in water supplies, aristolochicacid present in many medicinal plants and pioglitazone present in drugsfor the treatment of diabetes are associated as a risk factor (Poon etal., 2015; Rosenquist & Grollman, 2016).

According to the American Cancer Society, the reduced intake of liquidscan be a risk factor, since an individual who ingests high amounts ofliquids, mainly water, tends to eliminate chemicals more quickly, takinginto consideration that this will tend to deflate the bladder morefrequently (American Cancer Society, 2016).

In general, CB is about 3 to 4 times more common in men than in women(Nezos et al., 2009). On the other hand, women survival is worse withthis type of tumor. It is speculated that the high aggressiveness ofbladder cancer in women is due to hormonal imbalance, which arises fromthe fifth decade of life. Although the urinary bladder is secondarilyregulated by steroid sex hormones, the normal urothelial and tumorurothelial are responsive to androgens and estrogens (Garcia et al.,2015). Garcia et al. (2015) demonstrated for the first time in ratschemically induced to CBNMI that increased protein levels of ubiquitinligase SIAH-2 upregulated the androgenic receptors and decreased levelsof estrogen receptors, culminating in the escape of neoplasticurothelial cells from the immune system. These same authors found thatthe levels of immune system receptors, toll-like receptors (TLRs) weredecreased in CBNMI and associated this effect with the increase ofSIAH-2 levels and androgenic receptors.

The primary treatment of non-muscle invasive bladder cancer (NMIBC) isbased on surgical treatment through transurethral resection (RTU),followed by intravesical immunotherapy with Bacillus Calmette-Guerin(BCG), for decreasing recurrence and preventing tumor progression.However, the use of living and attenuated organisms can cause sideeffects and difficulty in predicting the immune and antitumor response.The use of BCG is limited in NMIBC-due to treatment failure, adverseeffects and intolerance occurring in more than two-thirds of patients.Although the use of RTU with chemotherapy or adjuvant immunotherapyrepresents an important breakthrough in the treatment of CBNMI, themanagement of this tumor, especially for high-grade tumors, remains achallenge due to the high recurrence rates and progression to invasiveand/or metastatic muscle phenotypes. The surgical option for such cases,partial or total cystectomy, is often associated with high rates ofmorbidity and mortality. Furthermore, for some patients, cystectomy isnot an available option due to the presence of concomitantcomorbidities. Thus, the development of new therapeutic modalities thatprevent disease progression, allow the preservation of the organ and thequality of life of patients and, finally, provide an option for thosewho are ineligible for cystectomy, is of utmost importance. Compoundsthat are able to act as agonists of the receptors of the immune system(toll-like receptors) can represent promising candidates to be developedas medicines against cancer.

In this context, the use of the biological response modifier-inorganicphosphate complex 1 (MRB-CFI-1) stands out, which has been proposed withpromising results in the treatment of NMIBC. Moreover, the invention ofthis new nanodrug for the treatment of CBNMI presents great efficiency,low toxicity and is economically viable, with great reproducibility andyield. After experiments with laboratory animals and clinical-veterinaryprotocol in dogs with CBNMI, the invention presents great potential foruse in humans.

BRIEF DESCRIPTION OF THE INVENTION

The present invention refers to a process of obtaining an inorganicnanostructured complex (CFI-1), a protein-associated nanostructuredcomplex (MRB-CFI-1) and use.

The nanostructured complex (CFI-1) comprises inorganic phosphate, with asize ranging from 190.0 to 310.3±36.4 nm, polydispersity of 0.563 andzeta potential of −22.6±4.15 mV.

The protein-associated nanostructured complex (MRB-CFI-1) comprisesprotein-associated phosphates, with a size ranging from 318.0 to477.1±146 nm, polydispersity of 0.9 and zeta potential of −28.60±6.74mV.

Further objects are the use of the complexes obtained (CFI-1) and(MRB-CFI-1) to treat cancer, preferably of prostate, bladder,colorectal, mastocytoma and lymphoma.

Although the compounds NH₄MgPO₄×6H₂O, (NH₄)₂MgH₂(PO₄)₂×4H₂O,(NH₄)₂Mg₃(HPO₄)₄×8H₂O and NH₄MgPO₄×H₂O are described in the literature,associated or not with hydrolytic proteins, they are objects to treatcancer in this invention. The present invention also discloses themechanisms of the aforementioned complexes in activating the immunesystem, both epithelial (local) and systemic against tumors.

BRIEF DESCRIPTION OF THE FIGURES

In order to achieve a full and complete view of the object of thisinvention, referenced figures are presented below, as follows.

FIG. 1 : X-ray diffraction pattern (XRD) of CFI-1.

FIG. 2 : Thermogravimetric analysis (TGA) of CFI-1 (A) and MRB-CFI-1(B).

FIG. 3 : Analysis of CFI-1 (A) and MRB-CFI-1 (B) differential scanningcalorimetry (DSC). Speed of 10° C./min.

FIG. 4 : Raman spectrum of CFI-1 (A) and MRB-CFI-1 (B).

FIG. 5 : Size and surface charge of CFI-1 (A) by dynamic lightscattering method (DLS) or photon correlation spectroscopy (PCS) byintensity. Zeta potential measured by electrophoretic mobility in ZetaSizer (MALVEM). (B) shows the same method applied to MRB-CFI-1.

FIG. 6 : MRB-CFI-1 (A) and CFI-1 (B) X-ray diffraction pattern. (C)Superposition of CFI-1 and MRB-CFI-1 standards.

FIG. 7 : Circular dichroism (CD) of CFI-1 and CFI-1 associated withP14-16 protein (or MRB-CFI-1).

FIG. 8 : Emission field scanning microscopies (FESEM micrograph) forCFI-1 (A) and MRB-CFI-1 (B), agree with the size measured by the dynamiclight scattering method (DLS) described in FIG. 5 .

FIG. 9 : Ultrasonography of the animals from control group (a) andinduced with MNU (b, c, d, e). (a) Urinary bladder morphology withnormal aspects (a), (b) Tumor masses infiltrating the cranial, ventraland dorsal walls of the bladder, measuring 0.32 cm×0.21 cm (b), 0.32cm×0.24 cm (c) and 0.27 cm×0.21 cm (d). Color Doppler mapping showingintense blood flow inside the tumor mass (e).

FIG. 10 : Photomicrographies of the urinary bladder, ureter and kidneyof rats in the control group (a, b, c); MRB-CFI1 20 (d, e, f); MRB-CFI150 (g, h, i); and MRB-CFI1 100 (j, k, l), (a) and (b) normal urothelialcomposed of 2-3 cell layers: one layer of basal cells (Bc), one layer ofintermediate cells (Ic), and one superficial layer composed of umbrellacells (UC). (c) and (f) Normal nephrons: glomeruli (GI), podocytes (Pd),proximal contorted tubule (Pct), distal contorted tubule (DCT), maculadensa (Md), urinary pole (Up) and vascular pole (Vp). (j) and (k) flaturothelial hyperplasia and intense inflammation (asterisks and arrows),both in the urinary bladder and in the ureter; blood vessel (Bv). (l)Intense inflammation (asterisks) in the nephrons. (d) and (e) Minimalinflammation (asterisks and arrows) both in the urinary bladder and inthe ureter, (g) and (h) moderate inflammation (asterisks and arrows) andflat urothelial hyperplasia in both the urinary bladder and the ureter,(i) moderate inflammation (asterisks) in the nephrons. a-l:GI—glomerulus, Lp—lamina propria, Ur—urothelium.

FIG. 11 : Photomicrographies of the urinary bladder and mouse kidney ofcontrol groups (A, B), MRB-CFI-1 20 (c, d), MRB-CFI1 50 (e, f) andMRB-CFI1 100 (g, h). (a) normal urinary bladder urothelium consisting of2-3 cell layers: one layer of basal cells (Bc), one layer ofintermediate cells (Ic) and one superficial layer composed of umbrellacells (Uc). (b) and (d) Normal nephrons: glomeruli (GI), podocytes (Pd),proximal contorted tubule (Pct), distal contorted tubule (DCT), maculadensa (Md), urinary pole (Up) and vascular pole (Vp). (c) Minimalinflammation (asterisks and arrows) in the urinary bladder, (e) and (g)moderate inflammation (asterisks and arrows) and flat urothelialhyperplasia in the urinary bladder, (f) and (h) moderate inflammation(asterisks) in the nephrons. a-h: GI—glomerulus, Lp—lamina propria,Ur—urothelium.

FIG. 12 : Photomicrographies of the urinary bladder and rabbit kidney ofcontrol groups (a, b), MRB-CFI-1 20 (c, d), MRB-CFI1 50 (e, f) andMRB-CFI1 100 (g, h). (a) and (c) Normal urinary bladder urotheliumconsisting of 2-3 cell layers: one layer of basal cells (Bc), one layerof intermediate cells (Ic) and one superficial layer composed ofumbrella cells (Uc). (b) and (d) Normal nephrons: glomeruli (GI),podocytes (Pd), proximal contorted tubule (Pct), distal contorted tubule(DCT), macula densa (Md), urinary pole (Up) and vascular pole (Vp). (e)and (g) Moderate inflammation (asterisks and arrows) and flat urothelialhyperplasia in the urinary bladder, (f) and (h) moderate inflammation(asterisks) in the nephrons. a-h: GI—glomerulus, Lp—lamina propria,Ur—urothelium.

FIG. 13 : Pictures of the abdominal cavity and macroscopic assessment ofthe peritoneum of rats from the control groups (a), CFI-1 (b) andMRB-CFI-1 (c, d), (a) normal abdominal cavity and peritoneum withoutsigns of inflammation, (b) peritoneum with moderate signs ofinflammation characterized by flushing, increased vascularization andhemorrhagic points (asterisks), as well as small clusters of phosphatecrystals (arrows), (c), (d) intense peritoneal inflammationcharacterized by increased flushing, vascularization and haemorrhagicpoints (asterisks), and increased deposits of phosphate crystals(arrows) in the abdominal cavity.

FIG. 14 : Photomicrographs of the urinary bladders of control group (A,B), MNU (c, d) and MNU+inorganic phosphate complex 1 (CFI-1) (e, f, g,h). (a), (b) Normal urothelium consisting of 2-3 layers: a layer ofbasal cells (closed arrow head), an intermediate cell layer (arrow), anda superficial or apical layer composed of umbrella cells (open arrowhead), (c), (d), (f) Invasive urothelial carcinoma (pT1): neoplasticcells arranged in small groups (asterisks) invading the connectivemucosa and focal squamous differentiation, (e) Flat carcinoma in situ(pTis), characterized by cell atypia: bulky nuclei with reducedcytoplasm and prominent nucleoli (arrows), (g), (h) flat hyperplasiacomposed of several cell layers in the urothelium, but with nocytological atypia. a-h: Lp—connective mucosa, M—own muscular layer,Ur—urothelium.

FIG. 15 : Photomicrographs of the urinary bladders from groupsMNU+P14-16 (a, b, c, d) and MNU+MRB-CFI1 (e, f, g, h). (a), (b), (g),(h) Flat hyperplasia consisting of several cell layers in theurothelium: basal cell layer, middle cell layer and superficial layer,but without cytological atypia. (c), (d) noninvasive urothelialcarcinoma (pTa) characterized by papillary lesions and urothelial cellswith disordered arrangement and with loss of polarity; mitotic figures(arrows), (e), (f) Normal urothelium consisting of 2-3 layers: a layerof basal cells (close arrow head), an intermediate layer of cells(arrow) and a superficial or apical layer composed of umbrella cells(open arrow head), a-h: Lp—connective mucosa, M—own muscular layer,Ur—urothelium.

FIG. 16 : Ultrasound representative of the urinary bladder of Dogs 1 and2 in the following times: before the first instillation (a, b), afterthe first instillation (c, d) and after 3 (e, f), 6 (g, h), 18 (i, j)and 22 (k, l) instillations of MRB-CFI-1.

FIG. 17 : Ultrasound representative of the urinary bladder of Dogs 3 and4 in the following times: before the first instillation (a, b), afterthe first instillation (c, d) and after 3 (e, f), 6 (g, h), 18 (i, j)and 22 (k, l) instillations of MRB-CFI-3.

FIG. 18 : Ultrasound representative of the urinary bladder of Dogs 5 and6 in the following times: before the first instillation (a, b), afterthe first instillation (c, d) and after 5 (e, f), 6 (g, h), 8 (i, j) and22 (k, l) instillations of MRB-CFI-5.

FIG. 19 : Cytotoxicity of MRB-CFI-1 and 5637 cells of grade II urinarybladder carcinoma. (A) The 5637 cells were plated in a cell density of2.0×10⁴ and treated with serial dilutions of the compounds (12.5 mg,6.25 mg, 3.13 mg, 1.56 mg and 0.39 mg) over 24 hours of incubation. Eachvalue represents the mean±standard deviation of three independentexperiments (n=3). Cell viability was normalized for untreated control.(B) Representative images of the MRB-CFI-1 compound by the calcein-AM/PIassay. The 5637 cells were plated in a cell density of 2.0×10⁴ andtreated with 12.5 mg, over 24 hours of incubation. The images wereobtained with DAPI, GFP and propidium iodide, magnification of 100× and2×2 stretching. Line: I—control group; II—12.5 mg MRB-CFI-1. Column:1—Hoesch 33342 (DAPI); 2—Calcein (GFP); 3—Propidium iodide (PI),4—Fusion of all channels. Bar scale: 300 pm.

FIG. 20 : X-ray diffraction (XRD): (A) CFI-1-PIBR-2017; obtained asdefined in the example of embodiment (I). (B) CFI-1+ethanolamine;obtained as defined in the example of embodiment (II). (C) Superpositionof the two XRD figures.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention refers to processes ofobtaining an inorganic nanostructured complex (CFI-1), aprotein-associated nanostructured complex (MRB-CFI-1) and antitumor use.

Two examples of embodiments related to obtaining the inorganicnanostructured complex (CFI-1) and the protein-associated nanostructuredcomplex (MRB-CFI-1) are described below.

Exemplary Embodiment (I)

The process of obtaining a nanostructured complex (CFI-1) by chemicalsynthesis comprises the following steps:

(a) Preparing dibasic ammonium phosphate [(NH₄)₂HPO₄] in situ in thepresence of ammonia and orthophosphoric acid, in a mechanicalhomogenizer with minimum power of 500 W (for example, of the UltraTurrax type), at a temperature between 25 and 55° C. for 20-30 min up toneutralization;

(b) Mixing two salts: Hexahydrate magnesium chloride, at 1-3% (by mass)and dibasic ammonium phosphate obtained in step (a) at 1-4% (by mass)between 22 and 30° C. and pH 5-7, in a mechanical homogenizer withminimum power of 500 W (e.g., of Ultra-Turrax type) in a variablerotation range between 7000 and 15000 rpm for 30-40 min;

(c) Applying a pressure level to the mixture obtained in step (b) in ahigh pressure homogenizer (e.g.; NIRO) with the homogenization valvewith variable pressure between 400 and 700 bar, preferably 600 bar, andin the second stage, the homogenizing valve with variable pressurebetween 50 and 70 bar, preferably 60 bar, for up to 1 to 3 cycles,preferably 2 cycles;

(d) Cooling the suspension obtained at step (c) in ice bath at atemperature range comprised between 0 and 20° C., preferably 15° C.;

(e) Precipitating; and

(f) Washing the crystals with distilled and sterile water and drying at30 to 40° C., preferably at 37° C. for 24 to 72 h, preferably 48 h.

After step (f), the CFI-1 crystals were dried and weighed, showing amass yield of 45-50%.

The nanostructured complex (CFI-1), obtained as defined in theembodiment example (I) (PRODUCT 1), comprises inorganic phosphate withsize varying from 190.0 to 310.3±36.4 nm, polydispersity of 0.563 andzeta potential of −22.6+4.5 mV.

The process of obtaining a protein-associated nanostructured complex(MRB-CFI-1) by chemical synthesis comprises the following steps:

(a) Preparing dibasic ammonium phosphate [(NH₄)₂HPO₄] in situ in thepresence of ammonia and orthophosphoric acid, in a mechanicalhomogenizer with minimum power of 500 W (for example, of the UltraTurrax type), at a temperature between 25 and 55° C. for 20-30 min up toneutralization;

(b) Mixing two salts: Hexahydrate magnesium chloride, at 1-3% (by mass)and dibasic ammonium phosphate obtained in step (a) at 1-4% (by mass)between 22 and 30° C. and pH 5-7, in a mechanical homogenizer in avariable rotation range between 7000 and 15000 rpm for 30-40 min;

(c) Adding protein in a concentration of 0.5-1.5% (mass/mass),preferably 1%, to the complex obtained in step (b), wherein theaforementioned protein comprises the hydrolytic proteins selected fromthe group comprised by chitinase of Bacillus Subtilis (14 kDa) andlysozyme from egg whites (14 kDa), preferably lysozyme, which haveimmunomodulatory activity.

(d) Applying a pressure level to the mixture obtained in step (c) in ahigh pressure homogenizer (NIRO) with the homogenization valve withvariable pressure between 400 and 700 bar, preferably 600 bar, and inthe second stage, the homogenizing valve with variable pressure between50 and 70 bar, preferably 60 bar, for up to 1 to 3 cycles, preferably 2cycles;

(e) Cooling the suspension obtained at step (c) in ice bath at atemperature range comprised between 0 and 20° C., preferably 15° C.;

(f) Precipitating; and

(g) Washing the crystals with distilled and sterile water and drying at30 to 40° C., preferably at 37° C. for 24 to 72 h, preferably 48 h.

The protein added in step (c) preferably comprises a concentration of0.7%.

The protein-associated nanostructured complex (MRB-CFI-1), obtained asdefined in the example of embodiment (I), said PRODUCT 2, comprisesinorganic phosphate associated with protein, with size varying from318.0 to 477.1±146 nm, polydispersity of 0.9 and zeta potential of−28.60±6.74 mV.

Exemplary Embodiment (II)

The process of obtaining a nanostructured complex (CFI-1) by chemicalsynthesis comprises the following steps:

(a) Preparing an ultrapure (99.99%) dibasic ammonium phosphate[(NH₄)₂HPO₄] solution (molar mass: 132.6 g/mol) with concentrationcomprised in the range of 1 and 4% (by mass), preferably 1%, diluted in1,000-2,000 mL of distilled water, under magnetic stirring withcontrolled speed and rotation between 200 and 400 rpm, preferably 300rpm, at temperature between 22° C. and 30° C. and pH 8.26-8.57 for 5minutes;

(b) Adding from 0.5 to 2.0% of an amine selected from the groupcomprised by monoethanolamine; diethanolamine and triethanolamine,preferably monoethanolamine (2-aminoethanol; C₂H₇NO; molar mass: 61.08g/mol), in the solution of ammonium phosphate dibasic [(NH₄)₂HPO₄]obtained in step (a) under stirring, with rotation between 200 and 400rpm, at a temperature range between 22° C. and 30° C., and pH between9.72 and 9.80 for 5 minutes, until completing homogenization.

(c) Maintaining the solution of ammonium phosphate dibasic [(NH₄)₂HPO₄]and monoethanolamine, obtained in step (b), under mechanical stirring,rotation between 200 and 400 rpm, at temperature between 22° C. and 30°C. and pH 9.72-9.80;

(d) Preparing an ultrapure (99.99%) solution of hexahydrate magnesiumchloride (MgCl₂.6H₂O) (molar mass: 203.3 g/mol), in a concentrationcomprised in the range of 1-3% (by mass), preferably 2%, under stirringwith rotation comprised between 200 and 400 rpm, preferably 300 rpm, atthe temperature range between 22 and 30° C., and pH between 7.38 and7.56, for 5 min until completing homogenization; the solution issubsequently transferred into a 200-600 mL separation funnel or to aTitrette® Bottletop Burette apparatus with appropriate volume;

(e) Slow and controlled drip of the denser liquid obtained in step (d)in the resulting solution obtained in step (b) under stirring, withrotation between 200 and 400 rpm, at temperature between 22° C. and 30°C., and pH 9.72-9.80;

(f) Maintaining the solution resulting from step (e) under stirring for2 hours with rotation between 200 and 400 rpm, at temperature between22° C. and 30° C., and pH 8.10-8.20 until complete dissolution;

(g) Cooling the suspension obtained at step (f) in ice bath at atemperature range comprised between 0 and 20° C., preferably 15° C.;

(h) Precipitating;

(i) Washing the CFI-1 crystals with distilled and sterile water, anddrying at 30 to 40° C., preferably at 37° C. for 24 to 72 h, preferably48 h.

Drip control in step (e) is performed at the speed between 26 and 33drops per minute, in a time interval between 3-4 hours.

The pH in step (e) is controlled so that 30 minutes after the start ofdripping, the pH is comprised between 9.12 and 9.32; 1 hour after thestart of dripping, the pH is comprised between 8.81 and 8.89; 2 hoursafter the start of dripping, the pH is comprised between 8.25 and 8.43;and 3 hours after the start of dripping, the pH is comprised between8.10 and 8.20; the pH must be kept constant throughout the reaction.

After step (i), the CFI-1 crystals were dried and weighed, showing amass yield of 98-100%.

The nanostructured complex (CFI-1) in the presence of a compound with anamine group that acts as pH stabilizer, obtained as defined in theembodiment of example (II), said PRODUCT 3, comprises inorganicphosphate with an average size of 449.6±116.6 nm, polydispersity of 0.55and zeta potential of −20.0±5.1 mV.

The process of obtaining a nanostructured complex (CFI-1) associatedwith protein (MRB-CFI-1) in the presence of a compound with an aminegroup acting as a pH stabilizer comprises the addition of the CFI-1,obtained as defined in the example of embodiment (II), to a solid stateprotein, at 1:1, 1:2, 1:3 and preferably 1:2 weight/weight ratios,wherein the aforementioned protein is selected from the group comprisedby P14-16, Bacillus subtilis chitinase (14 kDa) or egg white lysozyme(14 kDa), which are known to have immunomodulatory activities.

The protein-associated nanostructured complex P14-16 (MRB-CFI-1)comprises inorganic phosphate mixed with protein, by simple addition ofprotein crystals to CFI-1 at appropriate concentrations for the study ofbladder cancer: with a mean size of 509.6±92.6 nm and zeta potential of−26.3±6.7 mV.

Further objects are the use of the complexes obtained (CFI-1) and(MRB-CFI-1) to treat cancer, preferably of prostate, bladder,colorectal, mastocytoma and lymphoma. Additionally, the complexes(CFI-1) and (MRB-CFI-1) can be used as adjuvants to commercialchemotherapeutic drugs to treat prostate, bladder, colorectal,mastocytoma and lymphoma cancers.

Although the compounds NH₄MgPO₄×6H₂O, (NH₄)₂MgH₂(PO₄)₂×4H₂O,(NH₄)₂Mg₃(HPO₄)₄×8H₂O and NH₄MgPO₄×H₂O are described in the literature,associated or not with hydrolytic proteins, they are objects to treatcancer in this invention.

Results—Embodiment Example (I)

Characterization of the Nanostructured Magnesium and Ammonium PhosphateComplex (CFI-1):

Analysis of XFD shows the presence of ammonium, magnesium and phosphate:Table 1A (CFI-1) shows a ratio of phosphate to magnesium of 3.2 andshows only traces of metals, such as iron and calcium, and a value ofthe remaining structure, such as NH4+H2O (calculated by difference byweight of total mass). By this analysis, the approximate unit cell wouldbe (NH₄)₆Mg₃(PO₄)₄. P/Mg ratio=3.2. Table 1B (MRB-CFI-1) shows aphosphate to magnesium ratio of 2.86. By this analysis, the unit cell is(NH₄)₆Mg₃(PO₄)₄, without considering the organic part.

TABLE 1A Fluorescence X-ray analysis (XFD) of CFI-1: Analyte Results (%)PO₄ 55.06 Mg 16.88 Ca 0.03 Fe 0.01 NH₄ 27.68 Total 99.66

Note: The NH₄ value, in table 1A, shows a value associated with H₂O. Theminimum chemical formula of these values was: (NH₄)₆Mg₃(PO₄)₄. PO₄/Mgratio=3.2

TABLE 1B Fluorescence X-ray analysis (XFD) of MRB-CFI-1: Analyte Results(%) PO₄ 50.47 Mg 17.62 K 1.41 Na 0.29 Ca 0.19 Mn 0.08 Fe 0.01 Rb 0.01NH₄ 29.4 Total 99.98

Note: PO₄/Mg ratio=2.86. The minimum chemical formula of these valueswas: (NH₄)₆Mg₃(PO₄)₄, without considering the organic part.

Table 2 shows the components of the CFI-1 and MRB-CFI-1 complex on thesurface of the crystals, such as magnesium, nitrogen, phosphorus andoxygen, and total absence of carbon by XPS. Therefore, this techniqueshows, on the crystal surface, nitrogen (NH₄), phosphate and magnesiumas the only components of compound CFI-1. In the case of MRB-CFI-1, theprotein components are shown. Therefore, for CFI-1 it would beNO₆Mg₃(PO)₂ and for MRB-CFI-1 it would be C₁₄NO₈Mg₂(PO₄)₂.

TABLE 2 Analysis of CFI-1 and MRB-CFI-1 by X-ray photoelectronspectroscopy (XPS) (%) O Mg P N C Na K CFI-1 60.3 19.1 16.4 4.2 0 0 0MRB-CFI-1 45.6 6.8 11.4 3.2 31.3 1.3 0.6

The minimum chemical formula of the crystal surface was as follows: forCFI-1: NO₆Mg₃(PO₄)₂ and for MRB-CFI-1: C₁₄NO₈Mg₂(PO₄)₂.

The crystal surface has the following values: CFI-1 ratio: P/Mg=0.86;MRB-CFI-1 ratio=P/Mg=1.68.

X-Ray Refraction Pattern:

In FIG. 1 , the diffraction of CFI-1 is similar to some of the salts ofammonium and magnesium phosphates; however, they differ in terms ofintensities. Hence, they should have different distribution in the CFI-1complex. There is an increase in face expression (022) and decrease ofexpression in faces [002], [111] and [211] relative to other phosphatesalts. The phosphate/magnesium ratio of CFI-1 is 3.2, as demonstrated inthe previous analysis. Hence, the unitary cell of the CFI-1 clusterpresents a formula different from the other phosphate salts reported inthe literature, besides that the CFI-1 is nanoparticulated, as it isshown in the following item. Nanocrystallization evidently indicatesthat the mineralized product was oriented along a specific direction ofphases.

Fourier Transform Infrared Spectrum (FTIR):

The bands observed for CFI-1 (spectrum not shown in figure) around 3600,3500, 3260 and 3115 cm⁻¹, in the FTIR spectrum, probably belong to thestretching vibrations of group OH and the antisymmetric stretchingvibration of NH₄ groups. The water-PO₄—H bond appears around 2500 and2200 cm⁻¹. Water deformation appears at 1680 cm⁻¹ and the bands at 1600to 1400 cm⁻¹ were those of the deformation mode of the H—NH group ofNH₄. The PO₄ group alone is observed at 1006 cm⁻¹ (antisymmetricelongation), 571 cm⁻¹ (P—O flexion), 463 and 438 cm⁻¹ (PO₄ ⁻³ mode). At618 and 688 cm⁻¹ (Mg—O bond), and at 894 cm⁻¹ the deformation bond ofthe group with Mg). The water-water hydrogen bond was observed at 760and 695 cm⁻¹, while the bond between water hydrogen and NH₄ group wasobserved at 890 cm⁻¹. The MRB-CFI-1 FTIR spectrum presents the samebands with the addition of amide I 1640-1650 cm⁻¹ and amide II 1574-1550cm⁻¹ bands.

Thermogravimetric Analysis of CFI-1 and MRB-CFI-1:

FIG. 2A (CFI-1) shows weight loss at 100° C. of 8%, at 200° C. of 47%,at 250° C. of 50% and, at 350° C., loss of 53%. This shows loss ofammonia and water almost simultaneously. FIG. 2B showed weight loss at100° C., 150° C., 200° C., 250° C. and 350° C. of 25%, 48%, 52%, 53% and54%, respectively. Therefore, MRB-CFI-1 presented a faster weight lossthan CFI-1, probably due to the presence of protein.

Differential Scanning Calorimetry (DSC) Analysis of CFI-1 and MRB-CFI-1:

FIG. 3A (CFI-1) shows the temperature on set: 109.1° C., the enthalpy(J/g), (ΔH° m) 1.262, 00 J/g and melting point at 125, 94° C. for CFI-1.FIG. 3B (MRB-CFI-1) shows the temperature onset: 108.1° C., the enthalpy(J/g) (ΔH° m) 1,311.00 J/g and MP=122.37° C. MRB-CFI-1 presents amelting point lower than the CFI-1 due to the presence of the protein.

Raman Spectrum:

FIG. 4A shows the Raman bands of CFI-1 observed at 189 and 296 cm⁻¹,which are designated respectively as the MgO strain vibration and theO—Mg—O deformation vibration. The band at 574 cm⁻¹ can be assigned tothe v3 of PO₄ group. The 944 cm⁻¹ band can be assigned to thesymmetrical stretching band of P—O group. In the wide wavelength regionbetween 2670 and 3300 cm⁻¹ characteristics of vibration of strainvibration of H₂O and NH₄ ⁺ are found, and the small bands between 1400and 1740 cm⁻¹ correspond to their deformation vibrations. FIG. 4B showsMRB-CFI-1 and all of these bands appeared corresponding to phosphates,magnesium and ammonium groups, concomitant with conventional Raman bandsof amides between 1300-1650 cm⁻¹, corresponding to amides I, and III,respectively. Size (nm) and surface charge (zeta potential, mV) ofCFI-1:

FIG. 5A (CFI-1) shows a particle size value in the nano region of190.0-310.3±36.4 nm. The zeta potential measured was −22.6±4.15 mV. FIG.5B shows the MRB-CFI-1. Size ranging from 318.0 to 477.1±146 nm,polydispersity of 0.9 and zeta potential of −28.60±6.74 mV.

Solubility at Different pHs:

Table 3 shows the solubility of the CFI-1-water system that wasdetermined at 25 and 35° C. by means of crystal and solution balance ina container. An experimental solution of 100 ml of volume containing0.45 g of CFI-1 was treated at various pHs. The pH variation of thesolution was made by the addition of HCl and NaOH solutions. Themixtures were continuously stirred for 24 h to ensure the solutionsaturation. The undissolved solid was settled without agitation and,after 2 additional hours, it was filtered through a 0.22 μm membranefilter. The residue was dried overnight in the oven at 35° C. The drysamples were weighed using an analytical balance. The difference betweenthe residue and initial mass of CFI-1 provided the solubility. Table 3shows that the solubility value at pH 7 was 80 mg/I. This value canchange as a function of pH and ionic strength.

TABLE 3 Solubility of CFI-1 at different pHs [0.45 g/100 ml CFI-1].Solubility by weight difference (mg/L) pH 25° C. 35° C. 7.0 180 250 5.0270 285 3.0 300 325

Characterization of MRB-CFI-1 Complex:

X-Ray Diffraction Pattern (XRD).

FIG. 6 clearly shows the association of CFI-1 with lysozyme protein(P14-16). The superpositions of the two diffractions differ in some ofthe 2⊖ values. FIG. 5 shows that the product obtained in the presence ofP 14-16 protein had a higher expression of diffraction on faces (002)and (120), indicating that the mineralization product was preferablyoriented in a single direction. This shows a strong interaction of theprotein P14-16 in relation to CFI-1.

Circular Dichroism of MRB-CFI-1.

FIG. 7 shows in the UV 200-250 nm region, the magnitude of theellipticity at 208 nm and 222 nm, where there was a variation forMRB-CFI-1 complex at pH 7.4 (PBS). The increase indicated an increase inthe alpha-helix content of the native P14-16 protein (lysozyme). Thisincrease of alpha-helix content indicates a more orderly structure,since some of the residues should be involved in the interaction in thenon-helical region of the tertiary structure. This type of effect occursin proteins when they undergo the action of negative surfactants. Inthis case, the CFI-1, that is negative, shows the same effect.

Size (Nm) and Surface Charge (Zeta Potential, mV) of CFI-1 (A) Versus(B) MRB-CFI-1.

FIG. 8 showed a comparison of the different distribution when CFI-1 isassociated with P14-16 protein (MRB-CFI-1). MRB-CFI-1 (FIG. 8B) showslower homogeneity than CFI-1 alone (FIG. 8A). MRB-CFI-1 shows a smallincrease in particle size and surface charge (FIG. 5 ). Their analysesby FESEM clearly show the spherical and nanometric characteristics ofboth the CFI-1 (FIG. 8C) and MRB-CFI-1 (FIG. 8D).

In Vivo Toxicological and Biochemical Analyses of MRB-CFI-1:

For the toxicological and biochemical analyses of the nanodrugMRB-CFI-1, 20 Fischer 344 female rats, 20 C57BL/6 female mice and 20 NewZealand female rabbits were used.

The animals were distributed into 4 groups for each species, namely:control group (n=5 animals for each species): received an intravesicaldose of physiological solution 0.9%, for 6 consecutive weeks; groupMRB-CFI-1 20 (n=5 animals for each species): received an intravesicaldose of MRB-CFI-1 20 mg/Kg for 6 consecutive weeks; group MRB-CFI-1 50(n=5 animals for each species): received an intravesical dose ofMRB-CFI-1 50 mg/Kg for 6 consecutive weeks; group MRB-CFI-1 100 (n=5animals for each species): received an intravesical dose of MRB-CFI-1100 mg/Kg for 6 consecutive weeks.

The protocol for use of animals in research was approved by the EthicsCommittee on the Use of Animals (CEUA)-UNICAMP (protocols numbers:4536-1/2017; 4579-1/2017; 4435-1).

After the 6-week experimental period, all animals from each group wereeuthanized. For the local and systemic toxicity analyses of theMRB-CFI-1 nanodrug, the organs of the urinary system (urinary bladder,ureters and kidneys), and other target organs such as liver, spleen,stomach and pancreas, were collected and subjected to histopathologicalanalyses. The histopathology of these organs was evaluated and thetoxicity correlated with the degrees of inflammation. The degree ofinflammation was evaluated by a semi-quantitative scale: 0, absence ofinflammation, 1, minimal inflammation (less than five lymphocytes in anarea of 0.25 mm²), 2, moderate inflammation (mononuclear inflammatorycells scattered throughout the tissue, but still with visible stroma),3, intense inflammation (mononuclear inflammatory cells denselyinfiltrating the tissues.

Also, biochemical analyses were performed to verify the systemictoxicity of this compound, namely: alanine aminotransferase (ALT), aspecific marker for hepatic parenchymal lesion; aspartateaminotransferase (AST), a nonspecific marker for hepatic and/or cardiacinjury; alkaline phosphatase; as well as circulating levels ofcreatinine and urea to verify renal function. Spectrophotometricdeterminations were performed on a Pharmacia Biotech spectrophotometerwith a temperature-controlled cuvette chamber (UV/visible Ultrospec5,000 with Swift II application software for computer control, 97-4213,Cambridge, England, UK). All chemical reagents were from companyLaborLab (Guarulhos, Sao Paulo, Brazil).

In Vivo Assessment of Peritoneal Inflammatory Response afterAdministration of CFI-1, P14-16 Protein and MRB-CFI-1 Compound:

To verify whether compound MRB-CFI-1 and its constituents (CFI-1 andP14-16 protein) were able to deflate the peritoneal inflammatoryresponse (activation of the immune system) when administered directly tothe abdominal cavity, 8 7-week old Fischer 344 female rats were used,weighing 150 grams on average, which were obtained at the VivariumCenter of the State University of Campinas (CEMIB/UNICAMP).

The animals were divided into 4 groups (n=2 animals per group): Controlgroup: received an intraperitoneal dose of 0.3 mL of physiologicalsolution 0.9% every 72 hours, totaling 3 doses; group CFI-1: received anintraperitoneal dose of 20 mg/kg of CFI-1 suspended in physiologicalsolution 0.9% every 72 hours, totaling 3 doses; group P14-16: receivedan intraperitoneal dose of 20 mg/Kg of protein P14-16 suspended inphysiological solution 0.9% every 72 hours, totaling 3 doses; groupMRB-CFI-1: received an intraperitoneal dose of 20 mg/kg of compoundMRB-CFI-1 suspended in physiological solution 0.9% every 72 hours,totaling 3 doses. After 24 hours from the last application of eachcompound, the animals were euthanized and the peritoneums were evaluatedmacroscopically and collected for further histological assessment.

The protocol for use of animals in research was approved by the EthicsCommittee on the Use of Animals (CEUA)-UNICAMP (protocol number:4536-1/2017).

Pre-Clinical Trial: Induction and Treatment of Non-Muscle InvasiveUrinary Bladder Cancer (NMIBC) in Fischer 344 Rats:

In the present invention, 100 7-week Fischer 344 rats were used,weighing 150 grams on average, which were obtained from the VivariumCenter of the State University of Campinas (CEMIB/UNICAMP). For NMIBCinduction, 80 animals were anaesthetized with xylazine hydrochloride 2%(5 mg/kg i.m.; Köig, Sao Paulo, Brazil) and ketamine hydrochloride 10%(60 mg/kg, i.m.; Fort Dodge, Iowa, USA), maintained in this state for 45minutes to avoid spontaneous urination and a dose of 1.5 mg/kg ofN-methyl-N-nitrosourea (MNU-Sigma, St. Louis, Mo., USA) dissolved in 0.3ml of sodium citrate (1M pH 6.0) was instilled every 15 days (weeks 0,2, 4 and 6), totaling 4 doses (Fávaro et al., 2014; Garcia et al.,2016). The other 20 animals that did not receive MNU were considered asthe control group.

Two weeks after the last MNU dose, the animals were submitted to anultrasound examination to evaluate tumor occurrence. Ultrasounds wereevaluated using a portable software-controlled ultrasound system with a10-5 MHz 38 mm linear transducer.

Ultrasound of the urinary bladder of the animals induced with MNU showedtumor masses infiltrating the cranial, ventral and dorsal walls of theorgan, measuring 0.32 cm×0.21 cm; 0.32 cm×0.24 cm; and 0.27 cm×0.21 cm(FIGS. 9 b, 9 c and 9 d ). Color Doppler mapping evidenced intense bloodflow inside the tumor masses, reinforcing the diagnosis of urothelialneoplasia (FIG. 9 e ).

After NMIBC induction with MNU, the animals were distributed into 5groups (20 animals per group): control group (group 1): received anintravesical dose of 0.3 ml of physiological solution 0.9% for 6consecutive weeks; group MNU (cancer, group 2): received the sametreatment as group 1; group MNU+CFI-1 (group 3): received anintravesical dose of 20 mg/kg of CFI-1 suspended in physiologicalsolution 0.9% for 6 consecutive weeks; group MNU+P14-16 (group 4):received an intravesical dose of 20 mg/kg of protein P14-16 suspended inphysiological solution 0.9% for 6 consecutive weeks; group MNU+MRB-CFI-1(group 5): received an intravesical dose of 20 mg/Kg of compoundMRB-CFI-1 suspended in physiological solution 0.9% for 6 consecutiveweeks.

The intravesical doses in the different experimental groups wereinstillated using a 20 gauge flexible catheter (Abocath, Sao Paulo,Brazil). The animals from all experimental groups received water and thesame solid diet ad libitum (Nuvilab, Colombo, PR, Brazil). After 16weeks of treatment, the animals were euthanized and the urinary bladderswere collected and subjected to histopathological andimmunohistochemical analyses. The protocol for use of animals inresearch was approved by the Ethics Committee on the Use of Animals(CEUA)-UNICAMP (protocol number: 4536-1/2017).

Histopathologic Analysis:

For histological analysis, samples of the urinary bladder from allanimals of each experimental group (n=20 animals per group) werecollected and fixed with Bouin for twelve hours. After fixation, tissueswere washed in ethyl alcohol 70%, with subsequent dehydration in agrowing series of alcohols. Subsequently, the fragments were clearedwith xylene for 2 hours and included in plastic polymers (ParaplastPlus, ST. Louis, Mo., USA). Subsequently, the materials were sectionedusing a Slee CUT5062 RM 2165 microtome (Slee Mainz, Mainz, Germany) with5 micrometer thickness, stained with hematoxylin-eosin and photographedusing DM2500 photomicroscope (Leica, Munich, Germany).

The diagnosis of urothelial lesions was classified according to thestaging proposed by the common understanding of the World HealthOrganization/International Society of Urological Pathology (Epstein etal., 1998).

Antigen Immunostaining: TLR4, TRIF, IRF3, INF-γ, TLR2, IKK-α, MyD88,IL-6 and TNF-α:

Samples from the urinary bladder of all animals of each experimentalgroup (n=20 animals per group), the same used for histopathologicalanalyses, were used for immunostaining. Then, cuts with 5 μm thicknessin the rotating microtome Slee CUT5062 RM 2165 (Slee Mainz, Mainz,Germany) were collected on salinized sections. The antigenic recoverywas performed by incubation of the sections in citrate buffer (pH 6.0)at 100° C. in a microwave, or by treatment with proteinase K, dependingon the characteristics of each antibody. The blockage of endogenousperoxidases was carried out with H₂O₂(methanol 0.3%) with subsequentincubation in a blocking solution with bovine serum albumin (BSA) 3%, inTBS-T buffer for 1 hour at room temperature. Subsequently, the antigensTLR4, TRIF, IRF3, INF-γ, TLR2, IKK-α, MyD88, IL-6 and TNF-α were locatedthrough the specific primary antibodies (table 4), diluted in BSA 1% andstored overnight at 4° C. The Advance™ HRP kit (Dako Cytomation Inc.,USA) was used for antigen detection according to the manufacturerinstructions. After washing with TBS-T buffer, the cuts were incubatedwith conjugated secondary HRP antibody from the Advance™ HRP kit for 40minutes, and were subsequently revealed with diaminobenzidine (DAB),counterstained with Harris hematoxylin and evaluated using a DM2500photomicroscope (Leica, Munich, Germany).

To evaluate the immunoreactivity intensity of the antigens, thepercentage of positive urothelial cells was examined in ten fields foreach antibody with a 400× magnitude. The staining intensity was gradedon a 0-3 scale, and expressed as 0 (absence of immunoreactivity), 0% ofpositive urothelial cells; 1 (weak immunoreactivity), 1-35% of positiveurothelial cells; 2 (moderate immunoreactivity), 36-70% of positiveurothelial cells; and 3 (intense immunoreactivity), >70% of positiveurothelial cells (Garcia et al., 2016).

TABLE 4 Characteristics of the primary antibody for immunostaining.Primary antibodies Host species Code Source TLR2 Rabbit ABBI- Abbiotec,USA (policlonal) 251110 TLR4 Rabbit ABBI- Abbiotec, USA (policlonal)251111 IKK-α Rabbit sc-7218 Santa Cruz (policlonal) Biotechnology, CA,USA MyD88 Rabbit ab38515 abcam, USA (policlonal) IL-6 Rabbit ab83339Abcam, EUA (policlonal) TNF-α Rabbit ab6671 abcam, USA (policlonal) TRIFRabbit ab13810 Abcam, Cambridge, (policlonal) MA, USA IRF3 Rabbitab25950 Abcam, Cambridge, (policlonal) MA, USA INF-γ Mouse 507802Biolegend, USA (monoclonal)

Statistical Analysis:

The histopathological and immunohistochemical analyses were evaluatedusing the ratio test. For these analyses, a 5% type I error wasconsidered statistically significant.

Pre-Clinical Trial: Induction and Treatment of Non-Muscle InvasiveUrinary Bladder Cancer (NMIBC) in C57BL/6 Mice:

In the present invention, 100 7-week old C57BL/6J female mice were used,weighing 40 grams on average, which were obtained from the VivariumCenter of the State University of Campinas (CEMIB/UNICAMP). The animalsfrom all experimental groups received water and the same solid diet adlibitum (Nuvilab, Colombo, PR, Brazil). For NMIBC induction, 80 animalswere anaesthetized with xylazine hydrochloride 2% (5 mg/kg i.m.; König,Sao Paulo, Brazil) and ketamine hydrochloride 10% (60 mg/kg, i.m.; FortDodge, Iowa, USA), maintained in this state for 45 minutes to avoidspontaneous urination and a dose of 1.5 mg/kg of N-methyl-N-nitrosourea(MNU-Sigma, St. Louis, Mo., USA) dissolved in sodium citrate (1M pH 6.0)was instilled every 15 days (weeks 0, 2, 4 and 6), totaling 3 doses(Fávaro et al., 2012). The other 20 animals that did not receive MNUwere considered as the control group.

One week after the last MNU dose, the animals were submitted toultrasound examination to evaluate tumor occurrence and weresubsequently divided into 5 groups (n=20 animals per group) for therespective treatments: a) control group (group 1): received aintravesical dose of 0.1 ml of physiological solution 0.9% for 6consecutive weeks; b) MNU Group (cancer): received the same treatment asgroup 1; c) MNU+CFI-1 group (group 3): received an intravesical dose of20 mg/Kg of inorganic phosphate (CFI-1) for 6 consecutive weeks; d)MNU+P14-16 group (group 4): received an intravesical dose of 20 mg/kg ofP14-16 protein for 6 consecutive weeks; e) MNU+MRB-CFI-1 group (group5): received an intravesical dose of 20 mg/kg of MRB-CFI-1 compound for6 consecutive weeks.

The intravesical doses in the different experimental groups wereinstillated using a 22 gauge flexible catheter (Abocath, Sao Paulo,Brazil). Urine was collected weekly and, after treatment, the animalswere euthanized and the urinary bladders were collected. The protocolfor use of animals in research was approved by the Ethics Committee onthe Use of Animals (CEUA)-UNICAMP (protocol number: 4579-1/2017).

Histopathologic Analysis:

For histological analysis, samples of the urinary bladder from allanimals of each experimental group (n=20 animals per group) werecollected and fixed with Bouin for twelve hours. After fixation, tissueswere washed in ethyl alcohol 70%, with subsequent dehydration in agrowing series of alcohols. Subsequently, the fragments were clearedwith xylol for 2 hours and included in plastic polymers (Paraplast Plus,ST. Louis, Mo., USA). Subsequently, the materials were sectioned using aSlee CUT5062 RM 2165 microtome (Slee Mainz, Mainz, Germany) with 5micrometer thickness, stained with hematoxylin-eosin and photographedusing DM2500 photomicroscope (Leica, Munich, Germany). The diagnosis ofurothelial lesions was classified according to the staging proposed bythe common understanding of the World Health Organization/InternationalSociety of Urological Pathology (Epstein et al., 1998).

Cell Viability Assays

Cell viability of the MRB-CFI-1 nanodrug and its constituents wasevaluated in grade II urinary bladder carcinoma cells (cell line 5637),with 24 hours of incubation. For this, two approaches (MTT andcalcein/propidium iodide) were used, with different chemical agents toincrease the robustness of the results and avoid artifacts. The 5637cells were plated in a cell density of 2.0×10⁴ and treated with serialdilutions of the compounds (12.5 mg, 6.25 mg, 3.13 mg, 1.56 mg and 0.39mg) over 24 hours of incubation.

Statistical Analyses:

The histopathological analyses were evaluated using the ratio test. Forthese analyses, a 5% type I error was considered statisticallysignificant.

Results:

In Vivo Toxicological and Biochemical Analyses of MRB-CFI-1:

Serum levels of ALT, AST, alkaline phosphatase, urea and creatinine inrats, mice and rabbits treated intravesically with MRB-CFI-1 at doses of20 mg/kg, 50 mg/kg and 100 mg/kg did not differ statistically from theirrespective controls (tables 5, 6 and 7), indicating that this compounddid not present systemic toxic effects.

The urinary bladder, ureter and kidneys from rats, mice and rabbits inthe control group did not present inflammation and histopathologicalalterations (FIGS. 10 a, 10 b, 10 c, 11 a, 11 b, 12 a and 12 b ; table5). Rats from MRB-CFI-1 20 group showed minimal inflammation in theurinary bladder (100.0%), ureters (80.0%) and kidneys (60.0%) (FIGS. 10d, 10 e, 10 f , table 5). Moderate inflammation was observed in theurinary bladder (80.0%), ureters (80.0%) and kidneys (60.0%) of ratsfrom MRB-CFI-1 50 group (FIGS. 10 g, 10 h, 10 i , table 5). Rats fromMRB-CFI-1 100 group showed intense inflammation in the urinary bladder(80.0%), ureters (80.0%) and kidneys (60.0%) (FIGS. 10 j, 10 k , 101,table 5). Rats from MRB-CFI-1 50 and MRB-CFI-1 100 groups presented flathyperplasia in the urinary bladder urothelium and ureters (FIGS. 10 g,10 h, 10 j and 10 k ).

Mice from MRB-CFI-1 20 group showed minimal inflammation in the urinarybladder (100.0%) and ureters (100.0%), and absence of inflammation inkidneys (FIGS. 11 c, 11 d , table 6). Moderate inflammation was observedin the urinary bladder (100.0%), ureters (00.0%) and kidneys (100.0%) ofmice from the MRB-CFI-1 50 and MRB-CFI-1 100 groups (FIGS. 11 e, 11 f,11 g, 11 h , table 6). Flat hyperplasia was observed in the urinarybladder urothelium and ureters from mice of the MRB-CFI-50 and MRB-CFI-1100 groups (FIGS. 11 e and 11 g ). Rabbits from MRB-CFI-1 50 andMRB-CFI-1 100 groups showed moderate inflammation in the urinary bladder(100.0%) and kidneys (00.0%), while the animals from MRB-CFI-1 20 groupdid not present inflammation nor histopathological alterations in theorgans of the urinary system (FIGS. 12 c, 12 d, 12 e, 12 f, 12 g, 12 h ,table 7). Flat hyperplasia was observed in the urinary bladderurothelium and ureters from rabbits of the MRB-CFI-50 and MRB-CFI-1 100groups (FIGS. 12 e and 12 g ).

Absence of inflammation and histopathological alterations were verifiedin the liver, spleen, stomach and pancreas of all animals from eachspecies (tables 8, 9, 10).

TABLE 5 Toxicological and biochemical parameters for rats. Alkaline ALTAST phosphatase Urea Creatinine Groups (U/L) (U/L) (U/L) (mg/dL) (mg/dL)Control 22.9 ± 3.2 a 85.4 + 5.3 a 176.7 ± 15.2 a 38.5 ± 2.2 a 0.55 ±0.03 a MRB-CFI-1 20 22.8 ± 4.1 a 75.6 ± 9.1 a 183.1 ± 13.4 a 40.2 ± 4.0a 0.43 ± 0.04 a MRB-CFI-1 50 26.5 ± 5.4 a 73.3 ± 9.1 a 178.6 ± 31.1 a40.0 ± 1.6 a 0.49 ± 0.09 a MRB-CFI-1 100  28.1 ± 10.4 a  73.8 ± 10.6 a194.2 + 14.1 a 40.8 ± 3.8 a 0.51 ± 0.03 a

Data expressed as mean±standard deviation (n=5 animals per group).

Two means followed by the same lowercase letter do not differstatistically, according to the Turkey test (P<0.05).

TABLE 6 Toxicological and biochemical parameters for mice. Alkaline ALTAST phosphatase Urea Creatinine Groups (U/L) (U/L) (U/L) (mg/dL) (mg/dL)Control 11.7 ± 3.1 a 92.4 ± 5.5 a 154.1 ± 9.3 a 36.0 + 7.1 a 0.27 + 0.04a MRB-CFI-1 20 13.4 ± 2.7 a 94.8 ± 5.5 a 179.7 ± 7.8 a 32.3 ± 4.9 a 0.23± 0.05 a MRB-CFI-1 50 14.1 + 2.7 a 93.7 + 5.5 a  185.3 ± 10.7 a 25.9 ±3.7 a 0.34 + 0.14 a MRB-CFI-1 100 13.7 ± 2.0 a 94.4 ± 5.4 a 187.5 ± 8.8a 26.6 ± 3.5 a 0.31 + 0.08 a

Data expressed as mean±standard deviation (n=5 animals per group).

Two means followed by the same lowercase letter do not differstatistically, according to the Turkey test (P<0.05).

TABLE 7 Toxicological and biochemical parameters for rabbits. AlkalineALT AST phosphatase Urea Creatinine Groups (U/L) (U/L) (U/L) (mg/dL)(mg/dL) Control 20.9 + 2.6 a 42.0 ± 1.4 a 129.0 ± 12.7 a 39.7 ± 4.7 a0.90 ± 0.06 a MRB-CFI-1 20 30.5 ± 2.1 a 47.0 ± 1.4 a 126.0 ± 9.9 a  43.0± 4.2 a 0.85 ± 0.15 a MRB-CFI-1 50 33.4 ± 0.6 a 41.0 ± 1.3 a 137.5 ±10.6 a 40.2 ± 5.4 a 0.94 ± 0.06 a MRB-CFI-1 100 35.7 ± 1.8 a 47.5 ± 2.1a 126.0 ± 19.8 a 44.0 ± 4.2 a 0.95 ± 0.08 a

Data expressed as mean±standard deviation (n=5 animals per group).

Two means followed by the same lowercase letter do not differstatistically, according to the Turkey test (P<0.05).

TABLE 8 Semiquantitative assessment of inflammation in the urinarybladder, ureters, kidneys, liver, spleen, pancreas and stomach for the 5rats of each experimental group. Groups Control MRB-CFI-1 MRB-CFI-1MRB-CFI-1 100 (n = 05) 20 (n = 05) 50 (n = 05) (n = 05) Organs 1 2 3 4 51 2 3 4 5 1 2 3 4 5 1 2 3 4 5 Bladder 0 0 0 0 0 0 1 1 1 1 2 2 2 1 2 3 33 3 2 Ureter 0 0 0 0 0 0 1 1 1 1 2 2 2 1 2 3 3 3 3 2 Kidney 0 0 0 0 0 01 1 1 0 2 2 2 1 1 3 3 3 2 2 Liver 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 Spleen 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Pancreas 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 Stomach 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0. absence of inflammation, 1. minimal inflammation (less than fivelymphocytes in an area of 0.25 mm²), 2. moderate inflammation(mononuclear inflammatory cells scattered throughout the tissue, butstill with visible stroma), 3. intense inflammation (mononuclearinflammatory cells densely infiltrating the tissues.

TABLE 9 Semiquantitative assessment of inflammation in the urinarybladder, ureters, kidneys, liver, spleen, pancreas and stomach for the 5mice of each experimental group. Groups Control MRB-CFI-1 MRB-CFI-1MRB-CFI-1 100 (n = 05) 20 (n = 05) 50 (n = 05) (n = 05) Organs 1 2 3 4 51 2 3 4 5 1 2 3 4 5 1 2 3 4 5 Bladder 0 0 0 0 0 1 1 1 1 1 2 2 2 2 2 2 22 2 2 Ureter 0 0 0 0 0 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 Kidney 0 0 0 0 0 00 0 0 0 2 2 2 2 2 2 2 2 2 2 Liver 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 Spleen 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Pancreas 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 Stomach 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0. absence of inflammation, 1. minimal inflammation (less than fivelymphocytes in an area of 0.25 mm²), 2. moderate inflammation(mononuclear inflammatory cells scattered throughout the tissue, butstill with visible stroma), 3. intense inflammation (mononuclearinflammatory cells densely infiltrating the tissues.

TABLE 10 Semiquantitative assessment of inflammation in the urinarybladder, ureters, kidneys, liver, spleen, pancreas and stomach for the 5rabbits of each experimental group. Groups Control MRB-CFI-1 MRB-CFI-1MRB-CFI-1 100 (n = 05) 20 (n = 05) 50 (n = 05) (n = 05) Organs 1 2 3 4 51 2 3 4 5 1 2 3 4 5 1 2 3 4 5 Bladder 0 0 0 0 0 1 1 1 1 1 2 2 2 2 2 2 22 2 2 Ureter 0 0 0 0 0 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 Kidney 0 0 0 0 0 00 0 0 0 2 2 2 2 2 2 2 2 2 2 Liver 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 Spleen 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Pancreas 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 Stomach 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0. absence of inflammation, 1. minimal inflammation (less than fivelymphocytes in an area of 0.25 mm²), 2. moderate inflammation(mononuclear inflammatory cells scattered throughout the tissue, butstill with visible stroma), 3. intense inflammation (mononuclearinflammatory cells densely infiltrating the tissues.

Assessment of Peritoneal Inflammatory Response after Administration ofCFI-1, P14-16 Protein and MRB-CFI-1 Compound:

The macroscopic analyses of the peritoneum disclosed that the animalsfrom control groups (FIG. 13 a ) and P14-16 showed no inflammatorysigns, as well as increased vascularization. All animals from groupCFI-1 (FIG. 13 b ) presented moderate signs of peritoneal inflammation,which were characterized by flushing, increased vascularization andhemorrhagic points, and small clusters of phosphate crystals in theabdominal cavity. Regarding MRB-CFI-1 group, all animals presentedintense peritoneal inflammation characterized by increased flushing,vascularization and hemorrhagic points (FIGS. 13 c and 13 d ). Inaddition, there was an increase of deposits of phosphate crystals in theabdominal cavity. Thus, the present macroscopic results disclosed thatcompound MRB-CFI-1 was able to induce inflammatory response, whichjustifies its evaluation as an immunomodulator in the experiments inanimals with non-muscle invasive bladder cancer (NMIBC) chemicallyinduced, associated with its minimal or absent toxic effects.

Histopathological Analysis: Treatment of Non-Muscle Invasive UrinaryBladder Cancer (NMIBC) in Fischer 344 Rats:

The urinary tract of the control group did not present microscopicalterations (FIGS. 14 a and 14 b ; table 11). The normal urothelium wascomposed of 2-3 layers: a layer of basal cells, an intermediate celllayer and a superficial or apical layer comprised by umbrella cells(FIGS. 14 a and 14 b ).

In contrast, the urinary tract of the MNU Group (cancer) showed drastichistopathological alterations, such as: invasive urothelial carcinoma(pT1) (FIGS. 14 c, 14 d ) and noninvasive urothelial carcinoma (pTa) in60% and 40% of the animals, respectively (table 11). The pT1 carcinoma(FIGS. 14 c, 14 d, 14 f ) was characterized by neoplastic cells groupedin small groups or cords invading the connective mucosa, numerousmitotic figures and pleomorphic cells with enlarged nuclei and focalsquamous differentiation.

The most frequent neoplastic lesions in the MNU group+inorganicphosphate complex were the flat carcinoma in situ (pTis) (FIG. 14 e )and the pT1 carcinoma (FIG. 14 f ), which occurred in 40% and 20% of theanimals, respectively (table 11). The other animals did not presentmalignant lesions, and 20% of them presented flat hyperplasia (FIGS. 14g, 14 h ; table 11) and 20% normal bladder morphology, indicating thatthis treatment promoted 40% of tumor regression (table 11). pTiscarcinoma (FIG. 14 e ) was characterized by a disorderly proliferationof urothelial cells in a flat urothelium, with accentuated cellularatypia characterized by bulky nuclei, reduction of cytoplasm andmultiple and prominent nucleoli.

The histopathological analyses of the animals from MNU+P14-16 group had40% of tumor regression, and 20% of them presented flat hyperplasia(FIGS. 15 a, 15 b ; table 11) and 20% normal bladder morphology (table11). The most frequent neoplastic lesions in this group were high-gradepTa carcinoma (FIGS. 15 c, 15 d ) and pT1 carcinoma, both in 20% of theanimals (table 11). Non-invasive pTa carcinoma was characterized bypapillary or non-papillary lesions, urothelial cells with disorderlyarrangement and with loss of polarity, hyperchromatic, pleomorphicnuclei and prominent nucleoli (FIGS. 15 c, 15 d ).

The microscopic aspects of the urinary bladder of MRB-CFI-1 group weresimilar to those found in the control group. Normal urothelium was foundin 40% of the animals (FIGS. 15 e, 15 f ; table 11). The most frequenthistopathological alteration in this group was flat hyperplasia (FIGS.15 g, 15 h ) that occurred in 40% of the animals (table 11), indicatingthat this compound promoted tumor regression in 80% of the animals. Flathyperplasia was characterized by thickening of the urothelium andabsence of cytological atypia (FIGS. 14 h, 15 a, 15 b, 15 g and 15 h ).pTis carcinoma occurred in 20% of the animals in this group (table 11).

TABLE 11 Percentage of histopathological alterations in the urinarybladder of rats from different experimental groups. MNU + MNU + MNU +MRB- Histo- Control MNU CFI-1 P14-16 CFI-1 pathology (n = 20) (n = 20)(n = 20) (n = 20) (n = 20) Normal 20 (100%)* — 4 (20%)  4 (20%) 8 (40%) Flat — — 4 (20%)  4 (20%) 8 (40%)* hyperplasia High-grade — — 8 (40%)* —4 (20%)  urothelial neoplasia - carcinoma in situ (pTis) Non-invasive —8 (40%)* — 8 (40%) — urothelial carcinoma (pTa) Invasive 12 (60%)  4(20%)  4 (20%) urothelial carcinoma (pT1) *Statistical significance(ratio test, P < 0.0001). Benign lesions: flat hyperplasia; Malignantlesions: pTis, pTa and pT1.

Antigen Immunostaining: TLR4, TRIF, IRF3, INF-γ, TLR2, IKK-α, MyD88,IL-6 and TNF-α: Treatment of Non-Muscle Invasive Urinary Bladder Cancer(NMIBC) in Fischer 344 Rats:

Immunostainings for MyD88 and IKK-α were significantly moderated in thecontrol, MNU+CFI-1, MNU+P14-16 and MRB-CFI-1 groups in relation to theMNU group, which presented weak immunoreactivity for these antigens(table 12). Also, the immunostaining for IL-6 and TNF-α weresignificantly moderate in the MNU group compared to the otherexperimental groups, indicating that the nanodrug MRB-CFI-1 and itsconstituents did not induce the pathway for producing inflammatorycytokines mediated by TLRs 2 and 4.

In contrast, the immunomarkers for TLR2, TLR4, TRIF, IRF3 and INF-γ weresignificantly intense in the urothelium of MRB-CFI-1 and control groupsin comparison with the other experimental groups (table 12), indicatingthat the MRB-CFI-1 nanodrug was able to stimulate the interferon pathwaymediated by TLRs 2 and 4. Also, the immunomarkers for these antigenswere moderated in the MNU+CFI-1 and MNU+P14-16 groups in relation to theMNU Group (table 12).

TABLE 12 Mean immunostaining intensity for the different antigens in theurinary bladder of control, MNU, MNU + CFI-1, MNU + P14-16 and MNU +MRB-CFI-1 groups. MNU + MNU + MNU + MRB- Antigen Control MNU CFI-1P14-16 CFI-1 groups (n = 20) (n = 20) (n = 20) (n = 20) (n = 20) TLR4 3(91.8%)* 1 (10.4%)  2 (53.1%)  2 (52.0%)  3 (88.7%)* TRIF 3 (80.5%)* 1(8.6%)  2 (50.2%)  2 (57.2%)  3 (90.4%)* IRF-3 3 (87.5%)* 1 (8.9%)  2(59.7%)  2 (61.8%)  3 (91.0%)* IFN-γ 3 (88.2%)* 1 (9.5%)  2 (60.2%)  2(64.9%)  3 (91.0%)* TLR2 3 (92.4%)* 1 (15.0%)  2 (51.0%)  2 (56.3%)  3(89.7%)* IKK-α 2 (47.6%)* 1 (20.3%)  2 (50.1%)* 2 (54.7%)* 2 (50.4%)*MyD88 2 (50.1%)* 1 (17.4%)  2 (47.3%)* 2 (49.1%)* 2 (48.8%)* IL-6 1(16.0%)* 2 (47.2%)* 1 (20.8%)  1 (24.0%)  1 (22.5%)  TNF-α 1 (18.5%)* 2(49.0%)* 1 (15.3%)  1 (17.1%)  1 (24.6%)  0, absence of reactivity; 1,weak immunoreactivity (1%-35% positive urothelial cells); 2, moderateimmunoreactivity (36%-70% positive urothelial cells); 3, intenseimmunoreactivity (>70% positive urothelial cells). *Statisticalsignificance (ratio test, P < 0.0001).

Histopathological Analysis—Non-Muscle Invasive Urinary Bladder Cancer(NMIBC) Treatment in C57BL/6 Mice:

The urinary tract of the control group did not present microscopicalterations (table 13). In contrast, the urinary tract of the MNU Group(cancer) showed drastic histopathological alterations, such as: pT1carcinoma, pTa carcinoma and pTis carcinoma in 20%, 20% and 60% of theanimals, respectively (table 13).

The most frequent neoplastic lesions in the MNU+CFI-1 group were pTiscarcinoma and pTa carcinoma, which occurred in 20% and 40% of theanimals, respectively (table 13). The other animals did not presentmalignant lesions, with 20% of them presenting flat hyperplasia and 20%a pre-malignant lesion called low-grade intraurothelial neoplasia (table13), indicating that this treatment promoted regression and inhibitedtumor progression in 40% of the animals.

TABLE 13 Percentage of histopathological alterations in the urinarybladder of mice from different experimental groups. MNU + MNU + MNU +MRB- Histo- Control MNU CFI-1 P14-16 CFI-1 pathology (n = 20) (n = 20)(n = 20) (n = 20) (n = 20) Normal 20 (100%)* — — — 8 (40%)  Flat — — 4(20%)* 4 (20%)* 4 (20%)* hyperplasia Low-grade — — 4 (20%)* — 4 (20%)*intra- urothelial neoplasia High-grade 12 (60%)* 4 (20%)  4 (20%)  4(20%)  intra- urothelial neoplasia - carcinoma in situ (pTis)Non-invasive — 4 (20%) 8 (40%)* 8 (40%)* urotelial carcinoma (pTa)Invasive —  4 (20%)* — 4 (20%)* — urothelial carcinoma (pT1)*Statistical significance (ratio test, P < 0.0001). Benign lesions: flathyperplasia; pre-malignant lesions: low-grade intraurothelial neoplasia;malignant lesions: pTis, pTa and pT1.

The histopathological analyses of the animals from group MNU+P14-16presented 20% of tumor regression, and 20% of them presented flathyperplasia (table 13). The most frequent neoplastic lesions in thisgroup were pTis, pTa and pT1 carcinomas, both in 20%, 40% and 60% of theanimals, respectively (table 13).

The microscopic aspects of the urinary bladder of MRB-CFI-1 group weresimilar to those found in the control group. Normal urothelium was foundin 40% of the animals (table 13). Benign lesions such as flathyperplasia, and pre-malignant lesions such as low-grade intraurothelialneoplasia, were found in 20% and 20% of the animals, respectively (table13), indicating that this treatment promoted regression and inhibitedtumor progression in 80% of the animals. The most frequent neoplasticlesion in this group was pTis carcinoma in 20% of the animals (table13).

Clinical-Veterinary Assay: Treatment of Spontaneous Urinary BladderCancer in Dogs:

Several animal models experimentally induced for bladder cancer havebeen established, including chemically induced tumors. Although suchanimal models are in use in the research of bladder cancer, animalmodels in which the disease occurs naturally, mimic as close as possibleto humans and may be useful to assess new therapies (Wu et al., 2006;Arantes-Rodrigues et al., 2013), including therapy with MRB-CFI-1.

Naturally occurring bladder cancer in dogs can provide an excellentmodel as it approaches human invasive bladder cancer, specificallyhigh-grade invasive urothelial carcinoma in terms of cell and molecularcharacteristics; biological behavior, including sites and frequency ofmetastases; and response to therapy (Knapp et al., 2014).

To this end, the effects of intravesical immunotherapy with MRB-CFI-1 inthe progression of bladder cancer are being evaluated in 20 dogs,attended at the veterinary clinic “Dr. Ronaldo Tizziani” (Campinas, SaoPaulo, Brazil). After the diagnosis of urothelial carcinoma and theconsent of the dog's owners, the treatment with MRB-CFI-1 was initiated.The protocol for use of dogs in research was approved by the EthicsCommittee on the Use of Animals (CEUA)-UNICAMP (protocol number:4481-1/2017).

Dogs received 25 mg of MRB-CFI-1 dissolved in 2.0 mL of physiologicalsaline solution 0.9% intravesically (probing) or by cystocentesis,depending on the conditions of access to the urinary bladder of eachdog. These animals received a weekly dose of MRB-CFI-1 for sixconsecutive weeks. For maintenance therapy, the animals received a doseof MRB-CFI-1 every 15 days for 6 months and a monthly dose for another 6months.

The therapeutic effects of MRB-CFI-1 were evaluated by ultrasound duringthe treatment cycle. Ultrasound evaluations were performed at thefollowing times: before the first instillation, after the firstinstillation and after 3, 6, 18 and 24 instillations of MRB-CFI-1.

Six dogs completed the full therapeutic regimen with MRB-CFI-1, while 12were in the maintenance phase and 2 in the induction phase. Thefollowing are the results of the 6 dogs, who completed the fulltherapeutic regimen: Dog 1: Dachshund breed, gender: female, age: 9years; Dog 2: undefined breed (SRD), gender: female, age: 16 years; Dog3: Dachshund breed, gender: female, age: 9 years; Dog 4: Teckel breed,Gender: female, age: 1 year; Dog 5: Lhasa Apso breed, gender: female,age: 13 years; Dog 5: Dachshund breed, gender: female, age: 12 years;Dog 6: Poodle breed, gender: female, age: 16 years.

Results:

Biochemical Analyses of Dogs with Urinary Bladder Cancer UndergoingTreatment with MRB-CFI-1:

Serum hemoglobin, leukocytes, platelets, hepatic function (ALT) andrenal function (urea and creatinine) analyses indicated that thecomplete treatment with MRB-CFI-1 (24 applications) was not toxic to the6 dogs, and many parameters such as hemoglobin, leukocytes and ALTreached normal values with the proposed treatment (table 14).

Therefore, these exams indicated that treatment with MRB-CFI-1 showed nosigns of systemic toxicity at the proposed therapeutic dose.

Ultrasound Analyses of Dogs with Urinary Bladder Cancer UndergoingTreatment with MRB-CFI-1:

a) Dog 1: before the first instillation of MRB-CFI-1, it was observedthe presence of tumor mass with irregular contours, mixed echogenicityand hyperechoic echotexture, measuring 3.08 cm×1.89 cm, and volume of5.75 cm³ (FIG. 16 a , table 15). After 6 instillations of MRB-CFI-1, thetumor mass reduced 56.52% of its volume in relation to the initialultrasound (FIGS. 16 c, 16 e, 16 g , table 15). At the end of 24instillations, the tumor mass reduced 79.30% of its volume (FIGS. 16 i,16 k , table 15). At the beginning of treatment, dog 1 presentedhematuria, which disappeared after the third application of MRB-CFI-1and did not relapse even after the last application (table 15).

b) Dog 2: before the first instillation of MRB-CFI-1, it was observedthe presence of tumor mass with irregular contours, mixed echogenicityand hyperechoic echotexture, measuring 3.42 cm×2.75 cm, and volume of13.53 cm³ (FIG. 16 b , table 15). After 6 instillations of MRB-CFI-1,the tumor mass reduced 88.17% of its volume in relation to the initialultrasound (FIGS. 16 d, 16 f, 16 h , table 15). At the end of 24instillations, the tumor mass reduced 93.93% of its volume (FIGS. 16 j ,161, table 15). At the beginning of treatment, dog 2 presentedhematuria, which disappeared after the third application of MRB-CFI-1and did not relapse even after the last application (table 15).

c) Dog 3: before the first instillation of MRB-CFI-1, it was observedthe presence of tumor mass with irregular contours, mixed echogenicityand hyperechoic echotexture, measuring 4.22 cm×2.60 cm, and volume of14.93 cm³ (FIG. 17 a , table 15). After 6 instillations of MRB-CFI-1,the tumor mass reduced 61.35% of its volume in relation to the initialultrasound (FIGS. 17 c, 17 e, 17 g , table 15). At the end of 24instillations, the tumor mass reduced 81.0% of its volume (FIGS. 17 i,17 k , table 15). At the beginning of treatment, Dog 3 presentedhematuria, which disappeared after the eighteenth application ofMRB-CFI-1 (Table 15), without relapse after the end of treatment.

d) Dog 4: before the first instillation of MRB-CFI-1, it was observedthe presence of tumor mass with irregular contours, mixed echogenicityand hyperechoic echotexture, measuring 3.91 cm×1.84 cm, and volume of6.92 cm³ (FIG. 17 b , table 15). After 6 instillations of MRB-CFI-1, thetumor mass reduced 48.84% of its volume in relation to the initialultrasound (FIGS. 17 d, 17 f, 17 h , table 15). At the end of 24instillations, the tumor mass reduced 82.22% of its volume (FIGS. 17 j,17 l , table 15). At the beginning of treatment, Dog 4 presentedhematuria, which disappeared after the eighteenth application ofMRB-CFI-1 (Table 15), without relapse after the end of treatment.

e) Dog 5: before the first instillation of MRB-CFI-1, it was observedthe presence of tumor mass with irregular contours, mixed echogenicityand hyperechoic echotexture, measuring 2.25 cm×1.86 cm, and volume of4.07 cm³ (FIG. 18 a , table 15). After 6 instillations of MRB-CFI-1, thetumor mass reduced 44.71% of its volume in relation to the initialultrasound (FIGS. 18 c, 18 e, 18 g , table 15). At the end of 24instillations, the tumor mass reduced 84.5% of its volume (FIGS. 18 i,18 k , table 15). At the beginning of treatment, dog 5 presentedhematuria, which disappeared only after the last application (table 15).

f) Dog 6: before the first instillation of MRB-CFI-1, it was observedthe presence of tumor mass with irregular contours, mixed echogenicityand hyperechoic echotexture, measuring 2.84 cm×2.73 cm, and volume of11.08 cm³ (FIG. 18 b , table 15). After 6 instillations of MRB-CFI-1,the tumor mass reduced 74.45% of its volume in relation to the initialultrasound (FIGS. 18 d, 18 f, 18 h , table 15). At the end of 24instillations, the tumor mass reduced 86.28% of its volume (FIGS. 18 j,18 l , table 15). Dog 6 did not present hematuria since the beginning oftreatment until the end (table 15).

Thus, these results indicated that intravesical immunotherapy withMRB-CFI-1 was effective in reducing and preventing the progression ofurothelial neoplastic lesions in spontaneous cancer of the canineurinary bladder.

Cell Viability Assays

Cell viability of MRB-CFI-1 and its components, CFI-1 and proteinP14-16, was 76.01%±12.39, 68.63%±9.47 and 75.71%±11.52, respectively,using the maximum concentration of 12.5 mg, as denoted in the MTTreduction assay (FIG. 19A).

FIG. 19B demonstrates the impact of treatment with MRB-CFI-1 on cellmembrane integrity. In addition, negative cells for calcein wereobserved, indicating loss of cell viability, as well as positive cellsfor propidium iodide (PI), indicating cell death. The results with 12.5mg of MRB-CFI-1 compound showed 75.25%±6.19 of positive calcein and25.50%±2.52 of positive PI for cell death. Therefore, all techniquesreported comparable dose-response ratio, and the nanocompound MRB-CFI-1showed low toxicity, as expected from drugs with immunomodulatoryproperties.

TABLE 14 Clinical evaluation of dogs before and after intravesicaltreatments with MRB-CFI-1. Biochemical parameters Hemo- Leuko- Creat-Therapeutical globin cytes Platelets ALT Urea inine scheme (g/dl) (mm³)(mm³) (U/L) (mg/dl) (mg/dL) Before 1^(st) 10.6 29.900   355.000  95.036.0 1.20 instillation Dog 1 after 1^(st) 11.8 16.300   400.000  88.051.0 0.85 instillation After 24^(th) 12.1 13.000   375.000  81.0 42.00.98 instillation Before 1^(st) 12.6 28.000   455.000 188.0 25.0 1.10instillation Dog 2 after 6^(th) 12.0 16.300   350.000 129.0 35.0 0.80instillation After 24^(th) 13.1 11.000   400.000  68.0 22.0 1.00instillation Before 1^(st) 12.0  7.900 1,100.000  46.0 39.0 1.16instillation Dog 3 after 6^(th), 13.9  8.600   759.000  58.0 45.0 0.94instillation After 24^(th) 14.7 12.900   300.000  41.0 50.0 0.90instillation Before 1^(st) 11.4 15.800   430.000  64.0 54.0 1.15instillation Dog 4 after 6^(th), 12.3 18.800   390.000  80.0 53.0 0.78instillation After 24^(th) 12.0 17.000   410.000  84.0 56.0 0.66instillation Before 1^(st) 12.7 10.500   260.000  33.0 31.0 0.62instillation Dog 5 after 6^(th), 12.1  9.600   334.000  50.0 40.0 1.15instillation After 24^(th) 12.5 11.200   420.000  45.0 52.0 0.77instillation Before 1^(st) 14.2  9.100   260.000  52.0 34.0 0.85instillation Dog 6 after 6^(st) 13.9  8.300   360.000  40.0 31.0 0.88instillation After 24^(th) 14.7  7.900   330.000  48.0 33.0 0.92instillation

Reference values: Hemoglobin: After 1 year (12-18 g/dl); Leukocytes:After 1 year (6,000-18,000/mm³); Platelets: 150,000-500,000/mm³; ALT:10-88 U/I; Urea: 15-65 mg/dl; Creatinine: 0.5-1.5 mg/dl.

TABLE 15 Ultrasound assessments of tumor size, volume and reductioncompared to the presence of red blood cells in dogs urine before andafter intravesical treatments with MRB-CFI-1. Urine Ultrasoundparameters of the para- tumor meter I Tumor Red reduc- blood TherapeuticLength Width Volume tion cells regimen (cm) (cm) (cm³) (%) (p/ml) DogBefore 1^(st) 3.08 1.89  5.75 —  50.000 1 instillation After 1^(st)instillation 2.19 2.09  5.00 13.04  30.000 After 3^(rd) 2.06 1.82  3.5737.91  6.000 instillation After 6^(st) instillation 1.60 1.87  2.5056.52  4.000 After 18^(th) 1.66 1.44  1.80 68.69  1.000 instillationAfter 24^(th) 1.61 1.19  1.19 79.30    200 instillation Dog Before1^(st) 3.42 2.75 13.53 —  70.000 2 instillation After 1^(st)instillation 3.08 1.99  6.38 52.84  10.000 After 3^(rd) 2.66 1.81  4.5666.29  3.000 instillation After 6^(th) instillation 1.54 1.41  1.6088.17    500 After 18^(th) 1.79 1.12  1.17 91.35    200 instillationAfter 24^(th) 1.94 0.90  0.82 93.93    300 instillation Dog Before1^(st) 4.22 2.60 14.93 — 120.000 3 instillation After 1^(st)instillation 3.68 2.11  8.57 42.59  90.000 After 3^(rd) 3.22 2.33  9.1538.71  75.000 instillation After 6^(th) instillation 3.19 1.86  5.7761.35  15.000 After 18^(th) 2.63 1.66  3.79 74.61  3.000 instillationAfter 24^(th) 2.58 1.45  2.83 81.00    900 instillation Dog Before1^(st) 3.91 1.84  6.92 — 960.000 4 instillation After 1^(st)instillation 3.48 1.92  6.71  3.03 150.000 After 3^(rd) 4.14 1.74  6.56 5.20  78.000 instillation After 6^(th) instillation 2.76 1.53  3.3848.84  24.000 After 18^(th) 1.96 1.31  1.76 74.56  6.000 instillationAfter 24^(th) 2.35 1.00  1.23 82.22  2.000 instillation Dog Before1^(st) 2.25 1.86  4.07 — 464.000 5 instillation After 1^(st)instillation 2.19 1.81  3.75  7.86 200.000 After 3^(rd) 1.96 1.85  3.5113.75  98.000 instillation After 6^(th) instillation 1.94 1.49  2.2544.71  50.000 After 18^(th) 1.61 1.55  2.02 50.36  12.000 instillationAfter 24^(th) 1.27 0.98  0.63 84.50  3.000 instillation Dog Before1^(st) 2.84 2.73 11.08 —  1.500 6 instillation After 1^(st) instillation2.57 2.64  9.12 17.68  1.000 After 3^(rd) 3.03 2.06  6.70 39.53  2.500instillation After 6^(th) instillation 2.34 1.52  2.83 74.45  1.500After 18^(th) 2.49 1.20  2.02 81.76  1.000 instillation After 24^(th)2.69 1.04  1.52 86.28  1.200 instillation

Erythrocytes Reference Value (Urine 1): Up to 6,000 p/Ml

Analysis of Therapeutic Adjutancy of Intravesical Immunotherapy withMRB-CFI-1 and Systemic Chemotherapy with Cisplatin in Non-MuscleInvasive Bladder Cancer (CBNMI)

The histopathological effects of intravesical immunotherapy withMRB-CFI-1 combined with systemic chemotherapy with systemic cisplatinwere verified in Fischer 344 female rats, chemically induced tonon-muscle invasive bladder cancer (CBNMI), as per the method alreadydescribed above. After inducing NMIBC with N-methyl-N-nitrosourea (MNU),the animals were distributed in four experimental groups (n=7 animalsper group): group 1 (cancer): received an intravesical dose of 0.2 mL ofphysiological solution 0.9% for 6 consecutive weeks; group 2(cancer+MRB-CFI-1): received an intravesical dose of 20 mg/kg ofcompound MRB-CFI-1 for 6 consecutive weeks; group 3 (cancer+cisplatin):received an intraperitoneal dose of 0.25 mg/kg cisplatin once a week for4 consecutive weeks; group 4 (cancer+MRB-CFI-1+cisplatin): receivedsimultaneous treatment with MRB-CFI-1 and cisplatin in the sameconcentrations and through the same administration pathways as groups 2and 3. The protocol for use of animals in research was approved by theEthics Committee on the Use of Animals (CEUA)-UNICAMP (protocol number:4324-1).

The results showed urothelial carcinoma with invasion of lamina propria(pT1) and papillary carcinoma (pTa) in 100% of animals in the cancergroup.

The animals treated systemically with cisplatin showed a decrease in theprogression of urothelial neoplastic lesions in 14.28% of the animals,which presented a benign lesion characterized by papillary hyperplasia(table 16). The most frequent neoplastic lesions in this group werecarcinoma in situ (pTis), pTa carcinoma and pT1 carcinoma in 14.28%,57.14% and 14.28% of the animals, respectively (table 16).

The animals treated with intravesical MRB-CFI-1 showed a 42.85% decreasein the progression of urothelial neoplastic lesions, which presentednormal urothelium (table 16). The most frequent neoplastic lesions inthis group were pTis carcinoma and pTa carcinoma in 28.57% and 28.57% ofthe animals, respectively (table 16).

The treatment combined with intravesical immunotherapy with MRB-CFI-1and systemic chemotherapy with cisplatin showed better histopathologicalrecovery of the cancer state and decreased progression of urothelialneoplastic lesions in 71.42% of the animals, of which 42.85% had normalurothelium and 28.57% had a benign lesion characterized by flathyperplasia (table 16). The most frequent neoplastic lesion in thisgroup was pTis carcinoma in 28.57% of the animals (table 16).

Thus, it can be concluded that the combination of intravesicalimmunotherapy with MRB-CFI-1 and systemic cisplatin may be considered avaluable option for the treatment of patients who do not respond tostandard treatment with BCG and/or who do not meet the criteria forearly cystectomy.

TABLE 16 Percentage of histopathological alterations in the urinarybladder of rats from different experimental groups. Group 1 Group 2Group 3 Group 4 Urothelial lesions (n = 7) (n = 7) (n = 7) (n = 7) Nolesion Normal — 42.85% — 42.85% urothelium (3)* (3)* Benign Flat — — —28.57% lesions hyperplasia (2)* Papilliferous — — 14.28% — hyperplasia(1) Malignant Carcinoma — 28.57% 14.28% — lesions in situ (Ptis) (2)*(1) Papillary 42.85% 28.57% 57.14% 28.57% carcinoma (3)* (2) (4)* (2)(pTa) Urothelial 57.14% — 14.28% — carcinoma (4)* (1) with laminapropria invasion (pT1) Group 1: cancer, group 2: cancer + MRB-CFI-1,group 3: cancer + cisplatin, group 4: cancer + cisplatin + MRB-CFI-1.*Statistical significance (ratio test, P < 0.0001).

Results—Exemplary Embodiments (I) and (II)

The characterization of CFI-1 with pH control in the absence (PIBR 102017 012768 0) (CFI-1-PIBR-2017) and the presence of monoethanolamineare as follows:

Elementary Analysis:

(A) CFI-1 (obtained as defined in the exemplary embodiment (1)): C(0.05%), H (7.02%), N (5.35%). (B) CFI-1 (obtained as defined in theexemplary embodiment (II)): C (0.08%), H (6.92%), N (5.33%).

Analysis of XPS or X-Ray-Excited Photoelectron Spectroscopy:

(A) CFI-1 (obtained as defined in the exemplary embodiment (1)): P(16.4%), Mg (19.1%), N (4.2%), O (60.3%), P/Mg ratio=0.9. (B) CFI-1(obtained as defined in the exemplary embodiment (II)): P (16.9%), Mg(16.7%), N (5.0%) and 0 (61.3%), P/Mg ratio=1.0.

Size (Nm) and Surface Charge (Zeta Potential, mV):

(A) CFI-1 (obtained as defined in the exemplary embodiment (1)): shows ananoparticle size value of 310.3, ±56.9 nm; and zeta potential of−21.8±5.8 mV. (B) CFI-1 (obtained as defined in the exemplary embodiment(II)): shows a nanoparticle size value of 449.6±116.6 nm; and zetapotential of −20.0±5.1 mV.

X-Ray Fluorescence Spectroscopic Analysis (XRF):

(A) CFI-1 (obtained as defined in the exemplary embodiment (1)): PO₄(55.06%), Mg (16.88%), NH₄ (27.68), PO₄/Mg ratio=3.36. (B) CFI-1(obtained as defined in the exemplary embodiment (II)): PO₄ (56.43%), Mg(17.61%), NH₄ (25.13), PO₄/Mg ratio=3.02.

X-Ray Diffraction Pattern (XRD)

FIG. 20 clearly shows that the CFI-1-PIBR-2017 (A), obtained as definedin the exemplary embodiment (1), presents the same characteristics ofcrystalline diffraction as the CFI-1+ethanolamine (B), obtained asdefined in the exemplary embodiment (II). FIG. 20C shows thesuperposition of the two XRD figures.

The invention claimed is:
 1. A process of obtaining a protein-associatednanostructured complex (MRB-CFI-1), by chemical synthesis comprising thesteps of: (a) Preparing ammonium phosphate dibasic [(NH₄)2HPO₄] in situin the presence of ammonia and orthophosphoric acid, in a mechanicalhomogenizer with a minimum power of 500 W, at a temperature between 25and 55° C. up to neutralization; (b) Mixing two salts: magnesiumchloride, at 1-3% (by mass) and the ammonium phosphate dibasic obtainedin step (a) at a concentration of 1-4% (by mass) and at a temperaturebetween 22 and 30° C. and pH 5-7, in a mechanical homogenizer in avariable rotation speed between 7000 and 15000 rpm to form a complex;(c) Adding protein in a concentration of 0.5 to 1.5% (mass/mass) to thecomplex obtained in step (b); (d) Applying a pressure level to themixture obtained in step (c) in a high pressure homogenizer with ahomogenization valve with variable pressure between 400 and 700 bar, andin the second stage, the homogenizing valve with variable pressurebetween 50 and 70 bar, for up to 1 to 3 cycles; (e) Cooling thesuspension obtained at step (c) to a temperature range comprised between0 and 20° C.; (f) Precipitating crystals from the suspension obtained instep (e); and (g) Washing the crystals obtained in step (f) withdistilled and sterile water and drying at a temperature between 30 and40° C. for a time interval between 24 and 72 hours.
 2. The process,according to claim 1, wherein step (a) is performed in an ultra-turraxmechanical homogenizer for a time between 20 and 30 minutes.
 3. Theprocess, according to claim 1, wherein step (b) is performed in anultra-turrax mechanical homogenizer for a time interval between 30 and40 minutes.
 4. The process, according to claim 1, wherein, in step (c),the concentration of the protein suspension is 1% (mass/mass).
 5. Theprocess, according to claim 1, wherein, in step (c), the protein isselected from the group of hydrolytic enzymes with immunomodulatingproperties of low molar mass.
 6. The process, according to claim 1,wherein the protein is chitinase from Bacillus subtilis (14 kDa) or eggwhite lysozyme (14 kDa).
 7. The process, according to claim 6, whereinthe protein is egg white lysozyme (14 kDa).
 8. A protein-associatednanostructured complex (MRB-CFI-1), obtained by the process described inclaim 1, comprising inorganic phosphate associated with protein, with asize of 318.0 to 477.1±146 nm, a polydispersity of 0.9, and a zetapotential of −28.60±6.74 mV.
 9. A method for treating cancer in asubject, the method comprising administering the complex (MRB-CF1-1) ofclaim
 8. 10. The method according to claim 9, wherein the cancer isprostate, bladder, colorectal, mastocytoma or lymphoma cancer.
 11. Themethod according to claim 9, wherein the complex is administered in apharmaceutically acceptable carrier.